The primary goal of the proposed experiments is to identify the nature of Cl minus-dependent glutamate binding to hippocampal membranes and to correlate changes in the number or affinity of the binding sites with changes in the responsiveness of hippocampal cells to synaptic stimulation or to bath-applied agonists of various glutamate receptors. Chloride-dependent glutamate binding has been found to be two- to fourfold increased after transient exposure of isolated membranes or whole slices to high concentrations of glutamate or tyrosyl-glutamate. The proposed experiments will test three different hypothesis to explain these findings: (i) Modulatory low-affinity binding sites for these ligands are converted into or otherwise control the state of high-affinity Cl minus-dependent binding sites. (ii) Glutamate receptors are converted to a desensitized, high-affinity state. (iii) Glutamate becomes sequestered and accumulated in membrane vesicles through a countertransport mechanism. One set of experiments will attempt to answer the question whether glutamate "binding" should be reinterpreted as glutamate sequestration in membrane vesicles. If the traditional interpretation as "binding" is confirmed, electrophysiological and Na+ flux studies on slices will be performed to determine if the induced increase in the number of binding sites correlates with a change in the synaptic field potential or with a change in the response to externally applied agonists of various glutamate receptors. This will hopefully allow us to identify the physiological role of the Cl minus-dependent glutamate binding site and to decide whether increases in glutamate binding after exposure to Ca2+ and high concentrations of glutamate plays a role in synaptic potentiation or rather represent a desensitization process. A number of questions concerning properties of glutamate binding cannot be satisfactorily resolved without solubilizing the binding proteins. The third experimental goal is to find detergents and methods to solubilize the membranes without interfering with or eliminating glutamate binding. Success in this project would eventually lead to the purification and characterization of the binding protein. The long term objective of this project is to understand the mechanism and the role of receptor regulation in governing synaptic strength in the forebrain.